There is a need for improved means to obtain or manipulate fatty acid compositions, from biosynthetic or natural plant sources. For example, novel oil products, improved sources of synthetic triacylglycerols (triglycerides), alternative sources of commercial oils, such as tropical oils (i.e., palm kernel and coconut oils), and plant oils found in trace amounts from natural sources are desired for a variety of industrial and food uses.
To this end, the triacylglycerol (TAG) biosynthesis system in plants and bacteria has been studied. In the cytoplasmic membranes of plant seed tissues which accumulate storage triglycerides ("oil"), fatty acyl groups at the sn-2 position of the triglyceride molecules are incorporated via action of the enzyme 1-acylglycerol-3-phosphate acyltransferase (E.C. 2.3.1.51), also known as lysophosphatidic acid acyltransferase, or LPAAT.
By inspection of the LPAAT activities in isolated membranes from seed tissues, it has been shown that LPAAT specificities vary from species to species in accordance with the kinds of fatty acyl groups found in the sn-2 positions of the respective storage oils. For example, in the seeds of Cuphea species, which accumulate oils containing medium-chain fatty acids, it is possible to demonstrate an LPAAT activity which will utilize medium-chain acyl-CoA and lysophosphatidic acid (LPA) substrates. In contrast, LPAAT activity from the membranes of rapeseed embryos, in which the oil contains fatty acids of longer chain length, uses these medium-chain substrates much less readily, and predominantly uses long-chain unsaturated fatty acids. Similarly the meadowfoam plant (Limnanthes alba) accumulates an oil containing erucic acid (22:1) in all three sn positions and has a seed LPAAT activity able to use 22:1-CoA and 22:1-LPA, whereas rapeseed, which does not accumulate these fatty acids, has little or no such 22:1-utilizing LPAAT.
Similar studies with the enzymes responsible for the sn-1 and sn-3 acylations show that they are much less selective with respect to the substrate chain lengths. Thus, for a specific storage triglyceride in a given plant, the types of fatty acyl groups found in the sn-2 position of the oil are determined primarily by the specificity of LPAAT with respect to its acyl-donor substrates, i.e. acyl-CoAs. In addition, the selectivity of the LPAAT towards the acyl-CoAs is also influenced by the nature of the acyl group already attached in the sn-1 position of the acceptor substrates, i.e. the 1-acylglycerol-3-phosphate or lysophosphatidic acid (LPA) molecules.
The characterization of lysophosphatidic acid acyltransferase (also known as LPAAT) is useful for the further study of plant FAS systems and for the development of novel and/or alternative oils sources. Studies of plant mechanisms may provide means to further enhance, control, modify or otherwise alter the total fatty acyl composition of triglycerides and oils. Furthermore, the elucidation of the factor(s) critical to the natural production of triglycerides in plants is desired, including the purification of such factors and the characterization of element(s) and/or co-factors which enhance the efficiency of the system. Of special interest are the nucleic acid sequences of genes encoding proteins which may be useful for applications in genetic engineering.
Literature
Published characterizations of acyltransferase specificities in rapeseed membranes report that acyl group discrimination occurs primarily at the sn-2 acylation (Oo et al., Plant Physiol. (1989) 91:1288-1295; Bernerth et al, Plant Sci. (1990) 67:21-28).
Coleman (Mol. Gen. Genet. (1992) 232:295-303) reports the characterization of an E. coli gene (plsC) encoding LPAAT. The E. coli LPAAT is capable of utilizing either acyl-CoA or acyl-ACP as the fatty acyl donor substrate.
Hares & Frentzen (Planta (1991) 185:124-131) report solubilization and partial purification of a long-chain preferring LPAAT from endoplasmic reticulum in pea shoots. The purported solubilization is based solely on the inability to sediment LPAAT activity by high-speed centrifugation.
Wolter et al. (Fat Sci. Technol. (1991) 93:288-290) report failed attempts to purify a Limnanthes douglasii acyltransferase catalyzing the acylation of erucic acid to the sn-2 position of the glycerol backbone, and propose hypothetical methods of gene isolation based on cDNA expression in microorganisms.
Nagiec et al. (J. Biol. Chem. (1993) 268:22156-22163) report the cloning of an SLCI (sphingolipid compensation) gene from yeast and report homology of the encoded protein to the LPAAT protein of E. coli.
Taylor et al. (in "Seed Oils for the Future", ed. Mackenzie & Taylor (1992) AOCS Press) report acylspecificities for 18:1-CoA and 22:1-CoA substrates for LPAATs from several plant species and discuss attempts to purify a B. napus LPAAT.
Slabas et al. (Ch. 5, pages 81-95 (1993) in Seed Storage Compounds: Biosynthesis, Interactions, and Manipulation, ed Shewry & Stobart, Clarendon Press) discuss attempts to purify plant LPAAT proteins and note that all attempts to purify LPAAT to homogeneity have failed. Attempts to clone a corn LPAAT gene by complementation of an E. coli mutation at plsC are also discussed.
Oo et al. (Plant Physiol. (1989) 91:1288-1295) report characterization of LPAAT specificities in membrane preparations of palm endosperm, maize scutellum, and rapeseed cotyledon.
Cao et al. (Plant Physiol. (1990) 94:1199-1206) report characterization of LPAAT activity in maturing seeds of meadowfoam, nasturtium, palm, castor, soybean, maize, and rapeseed. LPAAT activity was characterized with respect to 22:1 and 18:1 LPA and acyl-COA substrates.
Laurent and Huang (Plant Physiol. (1992) 99:1711-1715) report that LPAATs in palm and meadowfoam which are capable of transferring 12:0 and and 22:1 acyl-CoA substrates to the sn-2 position of LPA, are confined to the oil-accumulating seed tissues.
Bafor et al. (Phytochemistry (1990) 31:2973-2976) report substrate specificities of TAG biosynthesis enzymes, including LPAAT, from Cuphea procumbens and C. wrighti.
Bafor et al. (Biochem. J. (1990) 272:31-38) report results of studies on regulation of TAG biosynthesis in Cuphea lanceolata embryos. Results of assays for LPAAT activity in microsomal preparations from developing cotyledons are provided.
Frentzen et al. (Eur. J. Biochem. (1990) 187:389-402 report characterization of mitochondrial LPAAT activity in potato tubers and pea leaves.
Hanke & Frentzen at Congress on Plant Lipids, Paris, Jul. 1, 1994 reported the obtention of a meadowfoam 1030 bp clone encoding a potential protein of 31 kDa. No sequence was shown but they indicated a "substantial" similarity to E. coli plsC and that this match was better than putative yeast LPAAT. The clone was reportedly obtained from a developing seed cDNA library in complementation studies with an E. coli LPAAT mutant. It was also reported that their clone demonstrated a higher preference for 22:1 CoA than 18:1 CoA as the acyl donor and that northern analysis showed expression in meadowfoam embryo and not in leaves.
Brown & Slabas, at the 4th International Congress of Plant Molecular Biology, Amsterdam, Jun. 19, 1994, showed a partial amino acid sequence reported to be a maize LPAAT obtained using a maize embryo culture cDNA to complement the E. coli LPAAT mutation. The molecular weight of the protein was reported at about 45 kDa with homologies to E. coli plsC and the yeast AT. Also, see W094/13814, published Jun. 23, 1994, which gives a sequence identified as the cDNA sequence of maize 2-acyltransferase.